Piezo1/ERK signaling pathway is activated in aortic VSMCs of patients with aortic dissection or aneurysm

doi:10.21203/rs.3.rs-2437600/v1

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Piezo1/ERK signaling pathway is activated in aortic VSMCs of patients with aortic dissection or aneurysm

https://doi.org/10.21203/rs.3.rs-2437600/v1

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Background

Piezo1 in vascular smooth muscle cells(VSMCs) is sensitive to the change of hemodynamic pressure and play the specific biological roles. ERK may be the key potential downstream signal of Piezo1. However, it remains unknown whether Piezo1/ERK signaling can be activated in aortic VSMCs of patients with aortic dissection(AD) or aneurysm(AA).

Methods

Piezo1 and p-ERK protein expressions were compared between AD/AA human aortic samples and normal samples by western blotting analysis and Immunohistochemistry. In addition, the rat thoracic VSMCs were divided into control, Yoda1( Piezo1 agonist), sch772984(ERK inhibitor) and Yoda1 + sch772984 groups. The cell life activities, including cell proliferation, migration, apoptosis, autophagy and phenotypical switch were measured.

Results

The patients with AD/AA had higher blood pressure than that of donors. We found that the expressions of Piezo1 and p-ERK in aortic media of AD/AA were higher than normal samples. In vitro experiments showed that Piezo1 could induce cell proliferation and migration by evoking ERK signaling, and this effect can be blocked by sch772984.

Conclusions

Piezo1/ERK signaling pathway is significantly activated in aortic VSMCs of patients with AD/AA, which might participate in AD/AA via promoting VSMCs proliferation and migration. This study provides a new insight into the biological action of Piezo1/ERK signaling pathway in the pathogenesis of AD/AA.

Piezo1

VSMC

aortic dissection

aortic aneurysm

Aortic dissection (AD) and aortic aneurysm (AA) are fatal vascular diseases with some common pathophysiological characteristics and risk factors. AA is aortic dilatation caused by progressive weakening of aortic wall. AD is mostly described as the blood from the intimal tear flowing into the media because of the special structure of aortic wall with three layers[1]. Over 95% of thoracic AA are asymptomatic until the point of AD or rupture. Without treatment, the mortality rate of AD is as high as 50% within 2 days[2]. It usually requires surgical intervention, especially Stanford type A AD(AD originating from the ascending aorta) [3]. Out of many factors affecting AD/AA, one common factor is hypertension[4]. Mechanism studies have indicated TGF-beta, Notch, SIRT1 and some inflammatory factors including matrix metalloproteinases (MMPs) play important roles in AD/AA[5–8]. In addition, some non-coding RNAs, such as miRNAs, lncRNAs and circRNAs, are also significantly involved in the pathogenesis of AD/AA[9]. Despite much research, the complicated pathogenesis and development mechanisms of AD/AA are not fully understood[10].

Piezo1 is a relatively newly discovered mechanically activated non-selective cation channel in mammals[11]. Previous studies have indicated that Piezo1 is involved in disease progression of different systems, including the hematologic system, cardiovascular system, digestive system, urinary system and respiratory system[11–15]. A recent study has demonstrated that Piezo1 is a sensor of shear stress or hypertension and plays an essential role in the development of vascular biology[16]. However, the expression of Piezo1 in human AD/AA remains unknown.

Meanwhile, extracellular signal-regulated kinases (ERK) has been shown recently as a key downstream signaling protein of Piezo1[17]. The mechanism is that the cell stretching induces opening of the stretch-activated calcium channel Piezo1, and calcium influx activates the calcium-sensitive kinase ERK[18]. ERK can trigger a series of biological reactions, such as cell proliferation, differentiation and apoptosis[19, 20]. Furthermore, phosphorylated ERK (p-ERK) is an important participant in AD/AA progression, which can induce vascular smooth muscle cells (VSMCs) proliferation and migration in media[21]. But the reason for over expression of ERK in AD/AA is not entirely clear. Here, we hypothesized that Piezo1 caused ERK activation to effect cell life activities of VSMC, which was involved in AD/AA progression.

In this study, we found that Piezo1 and p-ERK were up-regulated in aortic media of human AD/AA. In order to clarify the function mechanism, we examined the changes of cell proliferation, migration, phenotypical switch, apoptosis, and autophagy of rat thoracic VSMCs by regulating Piezo1 and p-ERK signaling, which may ultimately serve as a potential treatment target for AD/AA.

Clinical samples

Human aortic tissue samples were obtained from the hypertensive patients with Stanford type A AD(n = 6) or Thoracic AA(n = 6), who were undergone surgical replacement of the ascending aorta. Normal human aortic samples were obtained from non-hypertensive organ donors (n = 6). The patients with diabetes, congenital cardiovascular disease, systemic lupus erythematosus, multiple nodular arteritis, genetic aortic diseases, such as Marfan syndrome, and the history of smoking and drinking were excluded. This study protocol was conducted by the Declaration of Helsinki and was approved by Ethics Committee of Affiliated Hospital of Qingdao University. The tissue samples were collected immediately after they were resected during the surgery. The clinical information of the patients is shown in Table 1.

Table 1

Patients characteristics.
	Control (n = 6)	AD (n = 6)	AA (n = 6)
Sex (female/male)	2/4	3/3	2/4
Age (Years)	43.17 ± 2.32	45.17 ± 3.31	46.35 ± 2.38
BMI (kg/m2)	24.6 ± 3.2	25.1 ± 3.7	25.2 ± 3.6
Hypertension, n, (%)	0 (0)	6(100) *	6(100) *
Systolic pressure (mmHg)	124.20 ± 7.19	167.70 ± 8.16*	165.32 ± 8.05*
Diastolic pressure (mmHg)	72.83 ± 7.46	86.17 ± 6.24*	88.56 ± 7.12*
Diabetes, n, (%)	0 (0)	0 (0)	0 (0)
Smoker, n, (%)	0 (0)	0 (0)	0 (0)
Alcohol consumption, n, (%)	0 (0)	0 (0)	0 (0)
Ascending aorta diameter (mm)	2.82 ± 0.64	5.03 ± 0.45*	6.23 ± 0.40*^#
Note: BMI, body mass index; AD: aortic dissection; AA: aortic aneurysm;
*VS. Control, p < 0.05; #AA VS. AD, p < 0.05

Cell Culture And Treatments

The experimental procedures were performed according to the guidelines for the care and use of animals established by Qingdao University. Cells were isolated from the thoracic aorta of male Sprague–Dawley rats by enzymatic digestion as previously described[22]. Briefly, aortas were harvested and perivascular fat was removed. Aortas were digested in 1 mg/ml collagenase II, 0.744 U/ml elastase and 1 mg/ml Soybean Trypsin inhibitor (all reagents from Worthington Biochemical Corp.) in Hank’s balanced salt solution for 10 min. After digestion, the adventitia was removed under a dissecting microscope and the endothelium was scraped off with a cotton swab and then washed with PBS. For SMC isolation, aortas were cut into 0.5-mm pieces and placed in the enzyme solution for 1.5 hours. Disaggregated VSMCs were then grown and maintained in 20% serum-containing media (DMEM/F12 (Gibco), FBS (Hyclone), 100 U/ml penicillin/streptomycin (Gibco)). VSMCs were identified by immunofluorescence with an anti-alpha smooth muscle cell (a-SMA) antibody (Abcam, America). VSMCs were used at passage 5 to 8 for secondary culture. VSMCs were serum starved for 24 hours before experiment. Piezo1 agonist, Yoda1(10uM, Selleck, America) was used to activate Piezo1 pathway, and sch772984(10uM, meilunbio, China)(ERK blocker) was used to inhibit the function of ERK.

Measuring Cell Proliferation By Cell Counting Kit-8 Assay

The proliferation of rat VSMCs was measured using a Cell Counting Kit-8 (CCK-8, Beyotime,China). Briefly, cells were seeded in 96-well plates at a density of 2 × 10³ cells/well, followed by different medium intervention: (1) 2% FBS medium, (2) 10uM Yoda1 + 2% FBS medium, (3) 10uM SCH772984 + 2% FBS medium, and (4) 10uM Yoda1 + 10uM SCH772984 + 2% FBS medium. Then CCK-8 reagent was added, and the cells were incubated at 37℃ for 1 h. Subsequently, absorbance was measured with microplate spectrophotometer at 450 nm.

Measuring Cell Proliferation By Edu Assay

EDU cell proliferation detection kit (Beyotime C0071, China) was used to detect the proliferation ability of VSMCs. In brief, the cells were incubated with EDU dye with concentration of 10 at 37℃ for 2 hours, then the culture medium containing EDU was sucked, fixed at room temperature with 4% paraformaldehyde for 15 minutes and washed with PBS containing 3% BSA (Solarbio, Beijing, China) for three times, and then incubated at room temperature with PBS containing 0.3% Triton X-100(BioFroxx, Guangzhou, China) for 15 minutes and washed three times. For the EDU click reaction, the cells were treated with Click reaction solution, incubated for 30 min at RT in the dark and washed three times. For nuclear staining, Hoechst 33342 was diluted with PBS in the ratio of 1:1000 and incubated at room temperature in the dark for 10 minutes. Finally, it was observed under fluorescence microscope.

Measuring Cell Migration By Wound Scratch Assay

The effects of Piezo1/ERK signal on VSMCs migration were evaluated by scratch assay. VSMCs were seeded in 6-well plates. After the confluence, the monolayer VSMCs was scratched by a straight line using a sterile pipette tip. Then, the cells were washed with PBS to remove cell debris. Then different medium was added: (1) 2% FBS medium, (2) 10uM Yoda1 + 2% FBS medium, (3) 10uM SCH772984 + 2% FBS medium, and (4) 10uM Yoda1 + 10uM SCH772984 + 2% FBS medium. Images were recorded 24h after the monolayers were scratched. Image J software was used to measure the migration area.

Western Blotting Analysis

The proteins of aortic media samples or VSMCs were separated, and then isolated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, America). The membranes were blocked with 5% skimmed milk powder for 2 hours and incubated with the primary antibodies, including piezo1 (Abcam, America), ERK (Zenbio, Chengdu, China), p-ERK (Zenbio, Chengdu, China), α-SMA (Abcam, America), SM22α (Proteintech, America), OPN, ( Proteintech, America), LC3 (Abclonal, China), and β-actin (Bimake, Houston, America) overnight at 4℃. After cleaning with TBST, the membranes were incubated with Goat Anti-rabbit HRP antibody (Bioss, Beijing, China) at room temperature for 1 hour and the signals were detected with the Omin-ECL ultra-sensitive chemiluminescence detection kit (EpiZyme, Shanghai, China). The results were analyzed by Image J program. Full unedited gel blots could been seen in Additional file 1 in Supplementary Information.

Immunohistochemistry

The aortic tissues were fixed in 4% paraformaldehyde solution for 12 hours. The soaked samples were embedded in paraffin and sliced into 4um slices. The sections were incubated with primary antibody against piezo1 (Abcam, America), p-ERK (Zenbio, Chengdu, China), α-SMA (Abcam, America) or Ki67(Abclonal, China) overnight at 4°C and then were incubated with biotinylated secondary antibody (Abcam, ab150077/ab150113, America) for 1 hour at room temperature. The result was analyzed using Image Pro-Plus v. 6.0 software (Bethesda, MD, USA).

Tdt Mediated Dutp Nick End Labeling (Tunel)analysis

TUNEL apoptosis Detection Kit (Alexa Fluor488) (Yeasen, Shanghai, China) was used to detect nuclear DNA breaks during late apoptosis. Apoptotic VSMCs were detected by TUNEL staining. For all quantifications, five equivalent sections from each group were randomly selected and six visual fields from each section were observed through fluorescence microscope (Nikon, Japan).

Haematoxylin Eosin (He) And Masson’s Trichrome Staining

The isolated aortic tissues were fixed with 4% paraformaldehyde solution for 12 hours. The soaked samples were embedded in paraffin and sliced into 4um slices. HE staining and Masson’s trichrome staining were performed according to the instructions. The general morphology and compositions of aortic wall were analyzed under fluorescence microscope (Nikon, Japan).

Statistical analysis

All data were expressed as the mean ± standard deviation (SD). GraphPad Prism 8.0 was used to analyze the statistics and comparisons between groups were performed using one-way ANOVA. P < 0.05 was considered to be statistically significant.

Patients characteristics

Human aortic wall samples from 6 Stanford type A AD patients (AD group) and 6 AA patients (AA group) undergoing ascending aorta replacement and 6 organ donors (control group) were collected. The clinical information of the patients is shown in Table 1. The systolic pressure and diastolic pressure were higher in AD/AA groups. Ascending aorta diameter of AA was larger than AD or control groups.

The Disordered Structure And Vsmcs State Of Aortic Wall In Patients With Ad/aa

HE staining revealed that the normal aortic wall was composed of three layers: intima, media and adventitia. In the media, VSMCs were neatly arranged with few scattered inflammatory cells. However, the media of AD/AA patients became thinner, and there were increased inflammatory cells and loss of VSMCs. Masson’s trichrome staining showed that the level of collagen deposition was higher in the media of AD/AA samples compared with control group (Fig. 1a). Immunohistochemistry showed that expression of 𝛼-SMA decreased significantly in the ascending aortic media of patients with AD/AA compared to that of normal controls (Fig. 1b). The result may be due to the apoptosis of VSMCs in media of AD aorta. Ki67 staining indicated the level of VSMC proliferation was higher in AA group than other groups (Fig. 1c). Through TUNEL staining, the levels of VSMC apoptosis in patients with AD/AA were higher compared with the control group (Fig. 1d). Above results manifest VSMC proliferation and apoptosis coexist in the media of AD/AA patients.

Piezo1 And P-erk Is Upregulated In Aortic Media Of Ad/aa

According to the results of western blotting and immunohistochemical staining, we found Piezo1 was up-regulated in the aortic media of AD/AA patients compared with normal control group (Fig. 2a and b). Similarly, the ratio of p-ERK/T-ERK was higher in AD/AA groups (Fig. 2a and c). The results revealed the up-regulation of Piezo1 and p-ERK in VSMCs in ascending aortic wall of patients with AD/AA.

Piezo1 Activates Erk Signaling In Vsmcs

In vitro experiment, the rat thoracic VSMCs were divided into control, Yoda1( Piezo1 agonist), sch772984(ERK inhibitor) and Yoda1 + sch772984 groups. The study revealed ERK pathway was obviously activated by Piezo1(Yoda1), while the effect was blocked by the ERK inhibitor (Fig. 3). The results confirmed Piezo1 could activate ERK signaling pathway in VSMC.

Piezo1/erk Signaling Induces Vsmcs Proliferation

From EDU and CCK8 assays, we found Piezo1/ERK signaling pathway could induce VSMC proliferation significantly. Meanwhile, when the ERK signaling was blocked, the role of piezo1 inducing cell proliferation was significantly attenuated (Fig. 4).

Piezo1/erk Signaling Induces Vsmcs Migration

To determine the effect of Piezo1/ERK signaling on VSMC migration, we performed a wound scratch assay by Yoda1 or sch772984. As shown in Fig. 5, Piezo1(Yoda1) could enhance the ability of migration. The rate of migratory cell was reduced on VSMC with Yoda1 + sch772984 compared with Yoda1 group.

The Role Of Piezo1/erk Signaling In Vsmc Apoptosis, Autophagy And Phenotypical Switch

We assessed the role of Piezo1/ERK signaling on the autophagy and apoptosis in VSMC. The results of TUNEL staining showed that Yoda1 or sch772984 has no effect on VSMC apoptosis rate (Fig. 6). According to western blotting analysis, we found the downregulation of LC3-II/I in AD/AA tissues (Fig. 7a). In cell experiment, the autophagy level was not affected by over-expression of Piezo1/ERK signaling. However, inhibition of ERK signaling by sch772984 increased the protein expression of LC3-II/I (Fig. 7b).

To explore the function of Piezo1/ERK signalin for phenotypical switch in VSMC, the protein expression levels of α-SMA, SM22α and OPN were detected by a western blot assay. The protein expression of α-SMA and SM22α were down-regulated in AD/AA tissues, while OPN was over-expression (Fig. 7a). In vitro experiment, we found activation of Piezo1/ERK signaling did not cause phenotypical switch. To the contrary, inhibition of ERK signaling remarkably decreased the protein expression of α-SMA and SM22α, and induced the protein expression of OPN (Fig. 7b).

These results indicate that activated Piezo1/ERK signaling has no significant effect on VSMCs apoptosis, autophagy and phenotypical switch.

Hypertension is one of the most common factors affecting AD/AA. To further study the effect of hypertension in AD/AA progress, we focused on Piezo1, a Ca²⁺-permeable non-selective cationic channels activated by physical force. The existing research results have shown the principal roles piezo1 plays in different pathological and physiological states of circulatory system: it can regulate uteroplacental blood flow, pulmonary remodeling and vascular development by detecting physical force[23–25]. However, there is no literature on the mechanism of Piezo1 in AD/AA. As AD/AA most often occurs in the ascending aorta, we choose type A AD and thoracic AA for research[26]. Our findings showed that Piezo1 is significantly up-regulated in VSMCs in aortic media of AD/AA patients with hypertension, compared with normal tissue. The result indicates that Piezo1 is an important mechano-sensor of VSMCs in ascending aorta. VSMCs are the main cellular component of the aortic wall and play a vital role in degenerating the aortic wall in response to increased hemodynamic pressure, which marks the initiation of pathological remodeling in AD/AA[27]. So piezo1 may be an important mechanochemical coupling medium in VSMCs, and the affects of it on the life activities of VSMCs need to be clarified.

Piezo1 could activate downstream ERK signaling processes in dental cells, and ERK phosphorylation was significantly inhibited by ruthenium red (RR), a Piezo1 blocker[17]. In cell stretching experiment, epithelia cells displayed a faster proliferative response through Piezo1 triggering ERK signaling, compared with the slower speed proliferative response associated with YAP and TAZ[18]. In this experiment, we firstly confirmed that piezo1 could activate ERK signaling in VSMCs of thoracic aorta.

ERK is recognized as a key messenger leading to VSMCs proliferation. Excessive proliferation of VSMCs in media reduces the stability of morphology and function of the aortic wall in AA or AD[28]. One study has demonstrated the more rapid proliferation of VSMCs from dissected aorta compared with VSMCs from normal aortic tissues. Meanwhile, the expression levels of genes associated with proliferation were elevated[29, 30]. Our study showed that the proliferation of VSMCs increased significantly after activating piezo1/ ERK signaling pathway. Accordingly, when ERK signaling was blocked, the levels of cell proliferation decreased significantly. The results indicated piezo1 induced VSMCs proliferation by ERK.

It has been proved that the proliferation and apoptosis of VSMCs run through the whole process of aortic injury and remodeling, and the imbalance of them leads to the formation and development of AD/AA[31–33]. VSMCs excessive proliferation often occurs in early stage of diseases, such as hypertension and atherosclerosis[34]. On the contrary, the AD/AA tissues reveal higher level of apoptosis, indicating VSMCs apoptosis plays a leading role in the advanced stage of the diseases. According to vitro study, we speculated that piezo1/ERK signaling pathway was involved in AD/AA early progression probably through promoting VSMCs proliferation, rather than inducing apoptosis. To clarify VSMC-mediated pathomechanisms underlying early and advanced stages of aortic wall weakening is conducive to prevent and stabilize of AD/AA[35].

Abnormal migration of VSMC is the major contributing factor for pathological vascular remodeling through undermining arterial system stability. We found piezo1/ ERK signaling could enhance the ability of VSMCs migration, which might be one initial factor of AD/AA. During AD/AA progression, the balance between contractile and synthetic VSMCs is shifted towards synthetic VSMC. The switch is characterized by VSMC specific contractile proteins reduction, such as smooth muscle 22 alpha (SM22α) and alpha smooth muscle actin (αSMA), and synthetic proteins elevation, such as OPN. Furthermore, synthetic VSMC has high migration capability[36]. In this study, we found Piezo1 had no effect on VSMC phenotypic switch.

The effect of VSMCs autophagy on AD/AA remains controversial. On the one hand, excessive autophagy may induce VSMCs apoptosis and phenotypic switch[37, 38]. On the other hand, appropriate autophagy may have a therapeutic effect on AD/AA[39, 40]. The autophagy level in AD/AA tissues was decreased. In vitro experiments, it was found that the autophagy level of VSMC did not change significantly after the activation of piezo1 pathway.

ERK can regulate a series of biological reactions, such as cell proliferation, migration, differentiation and apoptosis[19, 20, 41], in this study, we found that Piezo1/ERK pathway has a significant effect on the proliferation and migration of VSMCs.

In summary, we concluded that the involvement of Piezo1/ERK signaling pathway in AD/AA might be due to proliferation and migration of VSMCs (Fig. 8). Our results reflected the real feature of AD/AA and provided new insight into the pathogenesis of AD/AA.

The limitation of our study was lack of further research on the middle mechanism between Piezo1 and ERK through mechanical force experiment. Further studies will be continued to elucidate the mechanisms of Piezo1 in the future.

Our study is the first to show that Pioze1 is an important mechano-sensor of ascending aorta, which is up-regulated in human AD/AA ascending aorta media. Pioze1 may activate ERK signaling pathway to increase proliferation and migration of VSMCs to induce dysfunction of aortic wall, contributing to the pathogenesis of AD/AA.

VSMCs: Vascular smooth muscle cells; AD: Aortic dissection; AA: Aortic aneurysm; MMPs: Matrix metalloproteinases; ERK: Extracellular signal-regulated kinases; a-SMA: Anti-alpha smooth muscle cell; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TUNEL: TdT Mediated dUTP Nick End Labeling; HE: Haematoxylin eosin; SD: Standard deviation; RR: Ruthenium red; SM22α: Smooth muscle 22 alpha.

Acknowledgements

Not applicable.

Authors’ contributions

YSM, SCY and ZH designed and wrote the paper. ZH, CWX, ZYZ, ZH and HHY performed research. All authors have read and approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (31670989 and 81700247).

Availability of data and materials

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

This study protocol was conducted by the Declaration of Helsinki and was approved by Ethics Committee of Affiliated Hospital of Qingdao University. All the participates were able to provide written informed consent or have an authorized principal (immediate relatives) willing to provide consent on their behalf. All animal experiments were approved by the Ethics Committee of Qingdao University，and carried out in accordance with ARRIVE guidelines and the 2011 revised form of The Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.

Consent to publication

Not applicable.

Competing interests

All the authors declare that there are no conflicts of interest.

Author details

Department of Cardiovascular Surgery, Affiliated Hospital of Qingdao University, 16 Jiangsu Road, Qingdao 266000, Shandong Province, China
Qingdao University, 308 Ningxia Road, Qingdao 266071, Shandong Province, China
Qingdao Fuwai Cardiovascular hospital, 201 Nanjing Road, Qingdao 266034, Shandong Province, China
Organ transplantation center, Affiliated Hospital of Qingdao University, 16 Jiangsu Road, Qingdao 266000, Shandong Province, China

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Piezo1/ERK signaling pathway is activated in aortic VSMCs of patients with aortic dissection or aneurysm

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Clinical samples

Cell Culture And Treatments

Measuring Cell Proliferation By Cell Counting Kit-8 Assay

Measuring Cell Proliferation By Edu Assay

Measuring Cell Migration By Wound Scratch Assay

Western Blotting Analysis

Immunohistochemistry

Tdt Mediated Dutp Nick End Labeling (Tunel)analysis

Haematoxylin Eosin (He) And Masson’s Trichrome Staining

Statistical analysis

Results

Patients characteristics

The Disordered Structure And Vsmcs State Of Aortic Wall In Patients With Ad/aa

Piezo1 And P-erk Is Upregulated In Aortic Media Of Ad/aa

Piezo1 Activates Erk Signaling In Vsmcs

Piezo1/erk Signaling Induces Vsmcs Proliferation

Piezo1/erk Signaling Induces Vsmcs Migration

The Role Of Piezo1/erk Signaling In Vsmc Apoptosis, Autophagy And Phenotypical Switch

Discussion

Limitations

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1